The bacterial reaction center protein (RC) is an integral membrane protein that initiates the light-driven electron transfer reactions of photosynthesis. With the advent of a high-resolution crystal structure, the RC has emerged as a central biophysical paradigm for understanding structure/function relationships in proteins. A broad range of biochemical, spectroscopic, structural and theoretical techniques has been trained on the RC system, in an effort to answer a question of fundamental biological relevance: How does the RC separate electrical charges across a membrane bilayer with unitary quantum yield? The complete specification of the mechanism requires the study of a host of energy transfer, electron transfer, proton transfer and conformational equilibrium reactions. A multidisciplinary approach may yield the most meaningful insights. In this proposal, attention is focused on the earliest electron transfer reactions that occur on the femtosecond and picosecond time scales. The P.I. proposes to approach the following specific mechanistic questions: 1) What accounts for the unidirectionality of electron transfer in the context of a quasisymmetric protein structure? 2) How is the sequence of electron transfer events to be described? In particular, what is the role of Beta(L), the monomeric bacteriochlorophyll? What kinetic description of these events is appropriate: superexchange, two step, inhomogeneous distributions of RCs? Is the initially-prepared excited state vibrationally equilibrated prior to electron transfer? What other types of relaxation processes are important to the mechanism? 3) What are the molecular determinants of the ground state and excited state spectroscopic and redox properties? Does the protein play an active or passive role in the mechanism? 4) What theoretical description of these processes is appropriate? Is the nonadiabatic theory of electron transfer adequate or is some other formalism required? The principles that are to be elucidated by these studies may be generally applicable to other energy-transducing proteins. The P.I. proposes to use time-resolved and steady-state optical spectroscopic techniques to observe the formation and decay of intermediates in wild-type and selected mutant RCs. Time-resolved fluorescence and absorption experiment will be conducted over a wide- range of experimental conditions. Global kinetic analysis will be applied to the data in order to attempt an unbiased, possibly unique mechanistic interpretation. The P.I. will also make modifications to the mutagenesis system and select more RC mutants for physicochemical analysis. In collaboration with others, mutant structures will be determined by x-ray crystallography, ENDOR spectra will be obtained, and resonance Raman spectra will be measured.